Recombinant proteins are produced and marketed in numerous agricultural, industrial and pharmaceutical applications. The robust productions of large quantities of recombinant proteins are necessary to provide abundant amounts of protein for these commercial applications. Unlike small molecules which are efficiently produced through chemical synthesis, the production of proteins and polypeptides are most efficiently produced in living cells such as bacteria, plants, or mammalian cells.
The Pseudomonas fluorescens expression system has been developed for efficiently producing large quantities of recombinant proteins. This expression system provides significant advantages for the expression of heterologous genes as compared to other known cellular expression systems. The quality, stability, solubility, titer, and rapid delivery of recombinant proteins produced by the Pseudomonas fluorescens expression system are superior to other know cellular expression systems. Moreover, the Pseudomonas fluorescens expression system is used to express proteins that cannot be expressed in other systems. As such, the Pseudomonas fluorescens expression system is a preferred system for the efficient production of recombinant proteins, thereby resulting in reduced costs for the production and development of recombinant proteins.
Improvements to the Pseudomonas fluorescens expression system have been developed to refine the system for the production of recombinant proteins via the heterologous expression of genes. US Pat App No. 2005/0186666 describes an improved expression system for the production of recombinant polypeptides utilizing auxotrophic selectable markers. US Pat App No. 2006/0008877 describes an improved method for producing recombinant proteins using Sec-system secretion signal peptides for secretion of recombinant proteins and peptides. US Pat App No. 2005/0202544 and International Pat App No. 2006/133210 describe novel inducible-promoters for commercial Pseudomonas fluorescens fermentation systems. US Pat App No. 2006/0110747 describes a process for improving the production levels of recombinant proteins by comparing two genetic profiles of a cell that expresses a recombinant protein and modifying the cell to change the expression of a gene product that is upregulated in response to the recombinant protein expression. US Pat App No. 2009/0162898 describes improved copy number plasmids containing a deletion, insertion, or substitution in the replication control region. US Pat App No. 2009/0062143 describes ribosomal binding site sequences for optimal expression of a heterologous protein. Despite the development of these innovations to the Pseudomonas fluorescens expression system, there is still a need in the art for improvements which result in the production of large quantities of recombinant proteins by the expression of heterologous genes.
The subject disclosure provides a novel method for the codon optimization of a polynucleotide sequence which results in robust production of large quantities of recombinant proteins via the Pseudomonas fluorescens expression system.